Angiotensin converting enzyme (peptidyl dipeptide hydrolase, hereinafter referred to as ACE) occupies a central role in the physiology of hypertension. The enzyme is capable of converting the decapeptide angiotensin I, having the sequence AspArgValTyrIleHisProPheHisLeu to an octapeptide, angiotensin II by removal of the carboxyterminal HisLeu. The symbols for various chemical entities are explained in the following table:
TABLE I ______________________________________ Ala = L-alanine Arg = L-arginine Asp = L-aspartic acid Gln = L-glutamine &lt;Glu = pyro-L-glutamic acid Gly = glycine Hip = Hippuric acid (Benzoyl glycine) His = L-histidine Ile = L-isoleucine Leu = L-leucine Lys = L-lysine Phe = L-phenylalanine Pro = L-proline Ser = L-serine Trp = L-tryptophan Tyr = L-tyrosine Val = L-valine ACE = Angiotensin converting enzyme Bicine = N,N-bis (2-hydroxyethyl) glycine EDTA = Ethylene diamine tetraacetic acid Hepes = N-2-hydroxyethlpiperazine-N'-2- ethanesulfonic acid HPP = p-hydroxyphenylpropionyl ______________________________________
Angiotensin I is formed by the action of the enzyme renin, an endopeptidase found in kidney, other tissues and plasma, acting on renin substrate, a serum .alpha..sub.2 globulin.
Blood pressure is affected by certain peptides found in the blood. One of these, angiotensin II, is a powerful pressor (blood pressure elevating) agent. Another, bradykinin, a nonapeptide with the sequence ArgProProGlyPheSerProPhe Arg is a powerful depressor (blood pressure lowering) agent. In addition to a direct pressor effect, angiotensin II stimulates release of aldosterone which tends to elevate blood pressure by causing retention of extracellular salt and fluids. Angiotensin II is found in measurable amount in the blood of normal humans. However, it is found at elevated concentration in the blood of patients with renal hypertension.
The level of ACE activity is ordinarily in excess, in both normal and hypertensive humans, of the amount needed to maintain observed levels of angiotensin II. However, it has been found that significant blood pressure lowering is achieved in hypertensive patients by treatment with ACE inhibitors. (Gavras, H., Brunner, H.R., Laragh, J.H., Sealey, J.E., Gavras, I., and Vukovich, R.A., New Engl. J. Med 291, 817 (1974). The ability to measure variations in the ACE activity in patients under treatment with an ACE inhibitor is therefore of great clinical and research importance. In addition, elevated levels of ACE activity have been found to exist in cases of sarcoidosis and also in Gaucher's disease. In some cases of sarcoidosis, ACE levels may be more than two standard deviations above the normal mean. In Gaucher's disease, levels of enzyme activity may be 60 times higher than those of normals. The elevated blood level seen in active sarcoidosis may fall to the normal range when the disease undergoes spontaneous remission or when therapeutic benefit is achieved through treatment. An effective, simple and convenient assay for the activity of ACE in a serum sample is accordingly a highly desirable tool of great utility to the physician who must deal with this disease, which is not only difficult to diagnose but to monitor.
ACE is a peptidyldipeptide hydrolase. It catalyzes the hydrolysis of the penultimate peptide bond at the C-terminal end of a variety of acylated tripeptides and larger polypeptides having an unblocked carboxyl group. The peptide hydrolysis is represented diagrammatically as: R--A.sub.2 --A.sub.1 + H.sub.2 O .fwdarw. R--OH + H--A.sub.2 --A.sub.1, wherein A.sub.1 is an amino acid at the carboxyl terminus of the peptide, A.sub.2 is an amino acid linked to A.sub.1 by a peptide bond, R is an N-substituted amino acid linked to A.sub.2 by a peptide bond. The action of ACE results in hydrolytic cleavage of the penultimate peptide bond from the carboxyl-terminal end yielding as reaction products a dipeptide, HA.sub.2 A.sub.1, and a remnant, R--OH.
The reactivity of the enzyme varies markedly depending on the substrate. At least one type of peptide bond, having the nitrogen supplied by proline, is not hydrolyzed at all. The apparent Michaelis constant (Km) varies from substrate to substrate over several orders of magnitude. For general discussion of the kinetic parameters of enzyme catalyzed reactions, see Lehninger, A., Biochemistry, Worth Publishers, Inc., New York, 1970, pp. 153-157. Many peptides which are called inhibitors of the enzymatic conversion of angiotensin I to angiotensin II are in fact substrates having a lower Km than angiotensin I. Such peptides are more properly termed competitive substrates. Examples of competitive substrates include bradykinin, and the peptide BPP.sub.5 .alpha. (also called SQ20475) from snake venom, whose sequence is &lt;GluLysTrpAlaPro.
ACE requires chloride ions for activity with some but not all substrates and is inhibited by divalent cation binding agents such as EDTA. Such inhibition is due to binding of Zn.sup.++ at the active site of the enzyme.
For background references see: White, A., Handler, P., and Smith, Ed, Principles of Biochemistry, 5th ed., 1973, McGraw-Hill, New York, pp. 589-590, 939-940; Bakhle, Y.S., in Handbook of Experimental Pharmacology; I. H. page and F. M. Bumpus, eds., vol. 37, pp. 41-80, Springer Verlag, Berlin, 1974. Soffer, R., Ann. Rev. Biochem. 45, 73 (1976); Ondetti, M.A., et al., U.S. Pat. No. 3,832,337, patented Aug. 27, 1974. Erdos, E. G., Am. J. Med. 60, (6), 749 (1976).